PROJECT SUMMARY Esophageal squamous cell carcinoma (ESCC) is the deadliest of all human squamous cell carcinomas with alcohol as a major risk factor. The role of alcohol (ethanol) in the esophageal epithelial cell injury and ESCC pathobiology remains unknown. Ethanol metabolism produces acetaldehyde, a major human carcinogen. Amongst the target organs alcohol drinking may cause diseases, esophagus is unique because it is directly exposed to high concentrations of EtOH and acetaldehyde. Alcohol detoxification involves clearance of acetaldehyde via aldehyde dehydrogenase 2 (Aldh2), the mitochondrial enzyme which breaks down aldehydes. Aldh2 dysfunction increases cancer risk in individuals with polymorphic Aldh2 mutation (Aldh2E487K). In Aldh2-/- and Aldh2E487K mutant mice and Aldh2-depleted human esophageal epithelial cells, alcohol induces mitochondrial damage and oxidative stress with increased DNA adducts formation and DNA damage, suggesting that mitochondrial dysfunction and reactive oxygen species (ROS) may contribute to genetic instability in Aldh2 dysfunctional esophageal epithelial cells. Alcohol-induced oxidative stress is alleviated by autophagy, a cytoprotective mechanism which removes damaged cellular components including dysfunctional mitochondria. Moreover, alcohol stimulates ESCC tumor growth with increased tumor-initiating cells displaying high autophagy and high CD44 expression (CD44H). The long-term goal is to identify esophageal mucosal defense mechanisms that can be manipulated for prevention or therapy of ESCC. The overall objective in this proposal is to clarify how alcohol affects esophageal epithelial cells with Aldh2 dysfunction. This proposal utilizes genetically engineered mouse models, patients' biopsies, human cell lines with altered Aldh2 status via the CRISPR/Cas9-approach, xenograft serial transplantation assays and a novel 3D esophageal organoid system with ethanol exposure and pharmacological interventions as a comprehensive platform to define the mechanistic and functional role of Aldh2 in epithelial response to alcohol exposure. The central hypothesis is that Aldh2 limits alcohol-induced mitochondrial dysfunction, suppressing oxidative stress and ESCC tumor initiation and growth. This hypothesis has been formulated on the basis of strong preliminary data produced in the applicant's laboratory and will be tested by pursuing the following three interrelated Specific Aims: (1) To clarify how mitochondrial Aldh2 limits alcohol-induced esophageal epithelial cell injury; (2) To define how autophagy reduces alcohol-induced mitochondrial damage and oxidative stress; (3) To determine how Aldh2 influences alcohol-induced esophageal neoplastic characteristics. These innovative studies will reveal novel insight into the role of dysfunctional Aldh2 in alcohol-induced oxidative stress and esophageal epithelial cell injury as well as autophagy-mediated cytoprotection in the pathogenesis of ESCC and other alcoholic diseases, both benign and malignant.